Apo-AI/AII peptide derivatives

ABSTRACT

The present invention concerns therapeutic agents that mimic the activity of Apo-AI amphipathic helix peptide. In accordance with the present invention, the compounds of the invention comprise:  
     a. a Apo-AI amphipathic helix peptide or Apo-AI amphipathic helix peptide-mimetic domain, preferably the amino acid sequence of SEQ ID NO: 7, or sequences derived therefrom by phage display, RNA-peptide screening, or the other techniques mentioned above; and  
     b. a vehicle, such as a polymer (e.g., PEG or dextran) or an Fc domain, which is preferred;  
     wherein the vehicle, preferably an Fc domain, is covalently attached to the Apo-AI amphipathic helix peptide or Apo-AI amphipathic helix peptide-mimetic domain. The vehicle and the Apo-AI amphipathic helix peptide or Apo-AI amphipathic helix peptide-mimetic domain may be linked through the N- or C-terminus of the Apo-AI amphipathic helix peptide or Apo-AI amphipathic helix peptide-mimetic domain, as described further below. The preferred vehicle is an Fc domain, and the preferred Fc domain is an IgG Fc domain. Preferred Apo-AI amphipathic helix peptide or Apo-AI amphipathic helix peptide-mimetic domains comprise the amino acid sequences described in Table 1. Other Apo-AI amphipathic helix peptide or Apo-AI amphipathic helix peptide-mimetic domains can be generated by phage display, RNA-peptide screening and the other techniques mentioned herein.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/198,920, filed Apr. 21, 2000, which is herebyincorporated by reference.

BACKGROUND OF THE INVENTION

[0002] A need exists for recombinant or modified therapeutic agentshaving Apo-AI amphipathic helix peptide activity.

[0003] Recombinant and modified proteins are an emerging class oftherapeutic agents. Useful modifications of protein therapeutic agentsinclude combination with the “Fc” domain of an antibody and linkage topolymers such as polyethylene glycol (PEG) and dextran. Suchmodifications are discussed in detail in a patent application entitled,“Modified Peptides as Therapeutic Agents,” U.S. Ser. No. 09/428,082, PCTappl. no. WO 99/25044, which is hereby incorporated by reference in itsentirety.

[0004] A much different approach to development of therapeutic agents ispeptide library screening. The interaction of a protein ligand with itsreceptor often takes place at a relatively large interface. However, asdemonstrated for human growth hormone and its receptor, only a few keyresidues at the interface contribute to most of the binding energy.Clackson et al. (1995), Science 267: 383-6. The bulk of the proteinligand merely displays the binding epitopes in the right topology orserves functions unrelated to binding. Thus, molecules of only “peptide”length (2 to 40 amino acids) can bind to the receptor protein of a givenlarge protein ligand. Such peptides may mimic the bioactivity of thelarge protein ligand (“peptide agonists”) or, through competitivebinding, inhibit the bioactivity of the large protein ligand (“peptideantagonists”).

[0005] Phage display peptide libraries have emerged as a powerful methodin identifying such peptide agonists and antagonists. See, for example,Scott et al. (1990), Science 249: 386; Devlin et al. (1990), Science249: 404; U.S. Pat. No. 5,223,409, issued Jun. 29, 1993; U.S. Pat. No.5,733,731, issued Mar. 31, 1998; U.S. Pat. No. 5,498,530, issued Mar.12, 1996; U.S. Pat. No. 5,432,018, issued Jul. 11, 1995; U.S. Pat. No.5,338,665, issued Aug. 16, 1994; U.S. Pat. No. 5,922,545, issued Jul.13, 1999; WO 96/40987, published Dec. 19, 1996; and WO 98/15833,published Apr. 16, 1998 (each of which is incorporated by reference inits entirety). In such libraries, random peptide sequences are displayedby fusion with coat proteins of filamentous phage. Typically, thedisplayed peptides are affinity-eluted against an antibody-immobilizedextracellular domain of a receptor. The retained phages may be enrichedby successive rounds of affinity purification and repropagation. Thebest binding peptides may be sequenced to identify key residues withinone or more structurally related families of peptides. See, e.g., Cwirlaet al. (1997), Science 276: 1696-9, in which two distinct families wereidentified. The peptide sequences may also suggest which residues may besafely replaced by alanine scanning or by mutagenesis at the DNA level.Mutagenesis libraries may be created and screened to further optimizethe sequence of the best binders. Lowman (1997), Ann. Rev. Biophys.Biomol. Struct. 26: 401-24.

[0006] Structural analysis of protein-protein interaction may also beused to suggest peptides that mimic the binding activity of largeprotein ligands. In such an analysis, the crystal structure may suggestthe identity and relative orientation of critical residues of the largeprotein ligand, from which a peptide may be designed. See, e.g.,Takasaki et al. (1997), Nature Biotech. 15: 1266-70. These analyticalmethods may also be used to investigate the interaction between areceptor protein and peptides selected by phage display, which maysuggest further modification of the peptides to increase bindingaffinity.

[0007] Other methods compete with phage display in peptide research. Apeptide library can be fused to the carboxyl terminus of the lacrepressor and expressed in E. coli. Another E. coli-based method allowsdisplay on the cell's outer membrane by fusion with apeptidoglycan-associated lipoprotein (PAL). Hereinafter, these andrelated methods are collectively referred to as “E. coli display.” Inanother method, translation of random RNA is halted prior to ribosomerelease, resulting in a library of polypeptides with their associatedRNA still attached. Hereinafter, this and related methods arecollectively referred to as “ribosome display.” Other methods employpeptides linked to RNA; for example, PROfusion technology, Phylos, Inc.See, for example, Roberts & Szostak (1997), Proc. Natl. Acad. Sci. USA,94: 12297-303. Hereinafter, this and related methods are collectivelyreferred to as “RNA-peptide screening.” Chemically derived peptidelibraries have been developed in which peptides are immobilized onstable, non-biological materials, such as polyethylene rods orsolvent-permeable resins. Another chemically derived peptide libraryuses photolithography to scan peptides immobilized on glass slides.Hereinafter, these and related methods are collectively referred to as“chemical-peptide screening.” Chemical-peptide screening may beadvantageous in that it allows use of D-amino acids and other unnaturalanalogues, as well as non-peptide elements. Both biological and chemicalmethods are reviewed in Wells & Lowman (1992), Curr. Opin. Biotechnol.3: 355-62. Conceptually, one may discover peptide mimetics of anyprotein using phage display, RNA-peptide screening, and the othermethods mentioned above.

SUMMARY OF THE INVENTION

[0008] The present invention concerns therapeutic agents that haveactivity similar to Apo-AI amphipathic helix peptide but with betterpharmaceutical characteristics (e.g., half-life). In accordance with thepresent invention, such compounds comprise:

[0009] a) a Apo-AI amphipathic helix peptide or Apo-AI amphipathic helixpeptide-mimetic domain, preferably having the sequence of Apo-AIamphipathic helix peptide, or sequences derived therefrom by phagedisplay, RNA-peptide screening, or the other techniques mentioned above;and

[0010] b) a vehicle, such as a polymer (e.g., PEG or dextran) or an Fcdomain, which is preferred;

[0011] wherein the vehicle is covalently attached to the Apo-AIamphipathic helix peptide or Apo-AI amphipathic helix peptide-mimeticdomain. The vehicle and the Apo-AI amphipathic helix peptide or Apo-AIamphipathic helix peptide-mimetic domain may be linked through the N- orC-terminus of the Apo-AI amphipathic helix peptide or Apo-AI amphipathichelix peptide-mimetic domain, as described further below. The preferredvehicle is an Fc domain, and the preferred Fc domain is an IgG Fcdomain. Apo-AI amphipathic helix peptide-mimetic domains can begenerated by phage display, RNA-peptide screening and the othertechniques mentioned herein.

[0012] Further in accordance with the present invention is a process formaking therapeutic agents having Apo-AI amphipathic helix peptideactivity, which comprises:

[0013] a. selecting at least one peptide having Apo-AI amphipathic helixpeptide activity; and

[0014] b. covalently linking said peptide to a vehicle.

[0015] The preferred vehicle is an Fc domain. Step (a) is preferablycarried out by selection SEQ ID NO: 7 or a sequence randomized therefromfrom phage display, RNA-peptide screening, or the other techniquesmentioned herein.

[0016] The compounds of this invention may be prepared by standardsynthetic methods, recombinant DNA techniques, or any other methods ofpreparing peptides and fusion proteins. Compounds of this invention thatencompass non-peptide portions may be synthesized by standard organicchemistry reactions, in addition to standard peptide chemistry reactionswhen applicable.

[0017] The primary use contemplated for the compounds of this inventionis as therapeutic or prophylactic agents. The vehicle-linked peptide mayhave activity comparable to—or even greater than—the natural ligandmimicked by the peptide.

[0018] The compounds of this invention may be used for therapeutic orprophylactic purposes by formulating them with appropriatepharmaceutical carrier materials and administering an effective amountto a patient, such as a human (or other mammal) in need thereof. Otherrelated aspects are also included in the instant invention.

[0019] Numerous additional aspects and advantages of the presentinvention will become apparent upon consideration of the figures anddetailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURES

[0020]FIG. 1 shows exemplary Fc dimers that may be derived from an IgG1antibody. “Fc” in the figure represents any of the Fc variants withinthe meaning of “Fc domain” herein. “X¹” and “X²” represent peptides orlinker-peptide combinations as defined hereinafter. The specific dimersare as follows:

[0021] A, D: Single disulfide-bonded dimers. IgG1 antibodies typicallyhave two disulfide bonds at the hinge region between the constant andvariable domains. The Fc domain in FIGS. 2A and 2D may be formed bytruncation between the two disulfide bond sites or by substitution of acysteinyl residue with an unreactive residue (e.g., alanyl). In FIG. 2A,the Fc domain is linked at the amino terminus of the peptides; in 2D, atthe carboxyl terminus.

[0022] B, E: Doubly disulfide-bonded dimers. This Fc domain may beformed by truncation of the parent antibody to retain both cysteinylresidues in the Fc domain chains or by expression from a constructincluding a sequence encoding such an Fc domain. In FIG. 2B, the Fcdomain is linked at the amino terminus of the peptides; in 2E, at thecarboxyl terminus.

[0023] C, F: Noncovalent dimers. This Fc domain may be formed byelimination of the cysteinyl residues by either truncation orsubstitution. One may desire to eliminate the cysteinyl residues toavoid impurities formed by reaction of the cysteinyl residue withcysteinyl residues of other proteins present in the host cell. Thenoncovalent bonding of the Fc domains is sufficient to hold together thedimer. Other dimers may be formed by using Fc domains derived fromdifferent types of antibodies (e.g., IgG2, IgM).

[0024]FIG. 2 shows the structure of preferred compounds of the inventionthat feature tandem repeats of the pharmacologically active peptide.FIG. 2A shows a single chain molecule and may also represent the DNAconstruct for the molecule. FIG. 2B shows a dimer in which thelinker-peptide portion is present on only one chain of the dimer. FIG.2C shows a dimer having the peptide portion on both chains. The dimer ofFIG. 2C will form spontaneously in certain host cells upon expression ofa DNA construct encoding the single chain shown in FIG. 3A. In otherhost cells, the cells could be placed in conditions favoring formationof dimers or the dimers can be formed in vitro.

[0025]FIG. 3 shows exemplary nucleic acid and amino acid sequences (SEQID NOS: 1 and 2, respectively) of human IgG1 Fc that may be used in thisinvention.

DETAILED DESCRIPTION OF THE INVENTION

[0026] Definition of Terms

[0027] The terms used throughout this specification are defined asfollows, unless otherwise limited in specific instances.

[0028] The term “comprising” means that a compound may includeadditional amino acids on either or both of the N- or C-termini of thegiven sequence. Of course, these additional amino acids should notsignificantly interfere with the activity of the compound.

[0029] The term “acidic residue” refers to amino acid residues in D- orL-form having sidechains comprising acidic groups. Exemplary acidicresidues include D and E.

[0030] The term “aromatic residue” refers to amino acid residues in D-or L-form having sidechains comprising aromatic groups. Exemplaryaromatic residues include F, Y, and W.

[0031] The term “basic residue” refers to amino acid residues in D- orL-form having sidechains comprising basic groups. Exemplary basicresidues include H, K, and R.

[0032] The term “hydrophilic residue” refers to amino acid residues inD- or L-form having sidechains comprising polar groups. Exemplaryhydrophilic residues include C, S, T, N, and Q.

[0033] The term “nonfunctional residue” refers to amino acid residues inD- or L-form having sidechains that lack acidic, basic, or aromaticgroups. Exemplary neutral amino acid residues include M, G, A, V, I, Land norleucine (Nle).

[0034] The term “vehicle” refers to a molecule that prevents degradationand/or increases half-life, reduces toxicity, reduces immunogenicity, orincreases biological activity of a therapeutic protein. Exemplaryvehicles include an Fc domain (which is preferred) as well as a linearpolymer (e.g., polyethylene glycol (PEG), polylysine, dextran, etc.); abranched-chain polymer (see, for example, U.S. Pat. No. 4,289,872 toDenkenwalter et al., issued Sep. 15, 1981; U.S. Pat. No. 5,229,490 toTam, issued Jul. 20, 1993; WO 93/21259 by Frechet et al., published Oct.28, 1993); a lipid; a cholesterol group (such as a steroid); acarbohydrate or oligosaccharide (e.g., dextran); or any natural orsynthetic protein, polypeptide or peptide that binds to a salvagereceptor. Vehicles are further described hereinafter.

[0035] The term “native Fc” refers to molecule or sequence comprisingthe sequence of a non-antigen-binding fragment resulting from digestionof whole antibody, whether in monomeric or multimeric form. The originalimmunoglobulin source of the native Fc is preferably of human origin andmay be any of the immnunoglobulins, although IgG1 and IgG2 arepreferred. Native Fc's are made up of monomeric polypeptides that may belinked into dimeric or multimeric forms by covalent (i.e., disulfidebonds) and non-covalent association. The number of intermoleculardisulfide bonds between monomeric subunits of native Fc molecules rangesfrom 1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g.,IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is adisulfide-bonded dimer resulting from papain digestion of an IgG (seeEllison et al. (1982), Nucleic Acids Res. 10: 4071-9). The term “nativeFc” as used herein is generic to the monomeric, dimeric, and multimericforms.

[0036] The term “Fc variant” refers to a molecule or sequence that ismodified from a native Fc but still comprises a binding site for thesalvage receptor, FcRn. International applications WO 97/34631(published Sep. 25, 1007 ) and WO 96/32478 describe exemplary Fcvariants, as well as interaction with the salvage receptor, and arehereby incorporated by reference in their entirety. Thus, the term “Fcvariant” comprises a molecule or sequence that is humanized from anon-human native Fc. Furthermore, a native Fc comprises sites that maybe removed because they provide structural features or biologicalactivity that are not required for the fusion molecules of the presentinvention. Thus, the term “Fc variant” comprises a molecule or sequencethat lacks one or more native Fc sites or residues that affect or areinvolved in (1) disulfide bond formation, (2) incompatibility with aselected host cell (3) N-terminal heterogeneity upon expression in aselected host cell, (4) glycosylation, (5) interaction with complement,(6) binding to an Fc receptor other than a salvage receptor, or (7)antibody-dependent cellular cytotoxicity (ADCC). Fc variants aredescribed in further detail hereinafter.

[0037] The term “Fc domain” encompasses native Fc and Fc variantmolecules and sequences as defined above. As with Fc variants and nativeFc's, the term “Fc domain” includes molecules in monomeric or multimericform, whether digested from whole antibody or produced by other means.

[0038] The term “multimer” as applied to Fc domains or moleculescomprising Fc domains refers to molecules having two or more polypeptidechains associated covalently, noncovalently, or by both covalent andnon-covalent interactions. IgG molecules typically form dimers; IgM,pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, ortetramers. Multimers may be formed by exploiting the sequence andresulting activity of the native Ig source of the Fc or by derivatizing(as defined below) such a native Fc.

[0039] The term “dimer” as applied to Fc domains or molecules comprisingFc domains refers to molecules having two polypeptide chains associatedcovalently or non-covalently. Thus, exemplary dimers within the scope ofthis invention are as shown in FIG. 2.

[0040] The terms “derivatizing” and “derivative” or “derivatized”comprise processes and resulting compounds respectively in which (1) thecompound has a cyclic portion; for example, cross-linking betweencysteinyl residues within the compound; (2) the compound is cross-linkedor has a cross-linking site; for example, the compound has a cysteinylresidue and thus forms cross-linked dimers in culture or in vivo; (3)one or more peptidyl linkage is replaced by a non-peptidyl linkage; (4)the N-terminus is replaced by —NRR¹, NRC(O)R¹, —NRC(O)OR¹, —NRS(O)₂R¹,—NHC(O)NHR, a succinimide group, or substituted or unsubstitutedbenzyloxycarbonyl-NH—, wherein R and R¹ and the ring substituents are asdefined hereinafter; (5) the C-terminus is replaced by —C(O)R² or —NR³R⁴wherein R², R³ and R⁴ are as defined hereinafter; and (6) compounds inwhich individual amino acid moieties are modified through treatment withagents capable of reacting with selected side chains or terminalresidues. Derivatives are further described hereinafter.

[0041] The term “peptide” refers to molecules of 3 to 40 amino acids,with molecules of 5 to 60 amino acids preferred. Exemplary peptides maycomprise the Apo-AI amphipathic helix peptide, peptides having one ormore residues of Apo-AI amphipathic helix peptide randomized, orpeptides comprising randomized sequences.

[0042] The term “randomized” as used to refer to peptide sequencesrefers to fully random sequences (e.g., selected by phage displaymethods or RNA-peptide screening) and sequences in which one or moreresidues of a naturally occurring molecule is replaced by an amino acidresidue not appearing in that position in the naturally occurringmolecule. Exemplary methods for identifying peptide sequences includephage display, E. coli display, ribosome display, RNA-peptide screening,chemical screening, and the like.

[0043] The term “Apo-AI amphipathic helix peptide-mimetic” refers to amolecule that increases or decreases one or more Apo-AI amphipathichelix peptide activity assay parameters as does Apo-AI amphipathic helixpeptide. An exemplary Apo-AI amphipathic helix peptide activity assay isdisclosed in Example 2.

[0044] The term “Apo-AI amphipathic helix peptide antagonist” refers toa molecule that increases or decreases one or more assay parametersopposite from the effect on those parameters by Apo-AI amphipathic helixpeptide. An exemplary Apo-AI amphipathic helix peptide activity assay isdisclosed in Example 2.

[0045] Additionally, physiologically acceptable salts of the compoundsof this invention are also encompassed herein. The term “physiologicallyacceptable salts” refers to any salts that are known or later discoveredto be pharmaceutically acceptable. Some specific examples are: acetate;trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide;sulfate; citrate; tartrate; glycolate; and oxalate.

[0046] Structure of Compounds

[0047] In General. Apo-AI amphipathic helix peptide binding amino acidsequences are described in Spinivas et al., 1990, Virology 176:48-57;Owens et al (1990), J. Clin. Invest. 86:1142-1150; Mendez et al. (1994),J. Clin. Invest. 94(4):1698-1705; Sprinivas et al. (1991), J. CellBiochem. 45(2):224-237. Each of these references is hereby incorporatedby reference in its entirety.

[0048] The present inventors identified particular preferred known ornaturally occurring sequences. These sequences can be randomized throughthe techniques mentioned above by which one or more amino acids may bechanged while maintaining or even improving the binding affinity of thepeptide.

[0049] In the compositions of matter prepared in accordance with thisinvention, the peptide may be attached to the vehicle through thepeptide's N-terminus or C-terminus. Thus, the vehicle-peptide moleculesof this invention may be described by the following formula I:

(A¹)_(a)-F¹-(A²)_(b)  I

[0050] wherein:

[0051] F¹ is a vehicle (preferably an Fc domain);

[0052] A¹ and A² are each independently selected from -(L¹)_(c)-P¹,-(L¹)_(c)-P¹-(L²)_(d)-P²,-(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³, and-(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³-(L⁴)_(f)-P⁴

[0053] P¹, P², P³, and P⁴ are each independently sequences of Apo-AIamphipathic helix peptide or Apo-AI amphipathic helix peptide-mimeticdomains;

[0054] L¹, L², L³, and L⁴ are each independently linkers; and

[0055] a, b, c, d, e, and f are each independently 0 or 1, provided thatat least one of a and b is 1.

[0056] Thus, compound I comprises preferred compounds of the formulae

A¹-F¹  II

[0057] and multimers thereof wherein F¹ is an Fc domain and is attachedat the C-terminus of A¹;

F¹-A²  III

[0058] and multimers thereof wherein F¹ is an Fc domain and is attachedat the N-terminus of A²;

F¹-(L¹)_(c)-P¹  IV

[0059] and multimers thereof wherein F¹ is an Fc domain and is attachedat the N-terminus of -(L¹)_(c)-P¹; and

F¹-(L¹)_(c)-P¹-(L²)_(d)-P²  V

[0060] and multimers thereof wherein F¹ is an Fc domain and is attachedat the N-terminus of -L¹-P¹-L²-P².

[0061] Peptides. Any number of peptides may be used in conjunction withthe present invention. Peptides may comprise part of the sequence ofnaturally occurring proteins, may be randomized sequences derived fromthe sequence of the naturally occurring proteins, or may be whollyrandomized sequences. Phage display and RNA-peptide screening, inparticular, are useful in generating peptides for use in the presentinvention.

[0062] A peptide sequence particularly of interest is of the formula

Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu Ala Phe(SEQ ID NO: 7)

[0063] Molecules of this invention incorporating these peptide sequencesmay be prepared by methods known in the art. Any of these peptides maybe linked in tandem (i.e., sequentially), with or without linkers, and afew tandem-linked examples are provided in Table 2. Any peptidecontaining a cysteinyl residue may be cross-linked with anotherCys-containing peptide, either or both of which may be linked to avehicle. Any peptide having more than one Cys residue may form anintrapeptide disulfide bond, as well. Any of these peptides may bederivatized as described hereinafter.

[0064] Additional useful peptide sequences may result from conservativeand/or non-conservative modifications of the amino acid sequences of SEQID NO: 7.

[0065] Conservative modifications will produce peptides havingfunctional and chemical characteristics similar to those of the peptidefrom which such modifications are made. In contrast, substantialmodifications in the functional and/or chemical characteristics of thepeptides may be accomplished by selecting substitutions in the aminoacid sequence that differ significantly in their effect on maintaining(a) the structure of the molecular backbone in the area of thesubstitution, for example, as a sheet or helical conformation, (b) thecharge or hydrophobicity of the molecule at the target site, or (c) thesize of the molecule.

[0066] For example, a “conservative amino acid substitution” may involvea substitution of a native amino acid residue with a nonnative residuesuch that there is little or no effect on the polarity or charge of theamino acid residue at that position. Furthermore, any native residue inthe polypeptide may also be substituted with alanine, as has beenpreviously described for “alanine scanning mutagenesis” (see, forexample, MacLennan et al., 1998, Acta Physiol. Scand. Suppl. 643:55-67;Sasaki et al., 1998, Adv. Biophys. 35:1-24, which discuss alaninescanning mutagenesis).

[0067] Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. For example, amino acidsubstitutions can be used to identify important residues of the peptidesequence, or to increase or decrease the affinity of the peptide orvehicle-peptide molecules (see preceding formulae) described herein.Exemplary amino acid substitutions are set forth in Table 1. TABLE 1Amino Acid Substitutions Original Exemplary Preferred ResiduesSubstitutions Substitutions Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln,Asn Lys Asn (N) Gln Gln Asp (D) Glu Glu Cys (C) Ser, Ala Ser Gln (Q) AsnAsn Glu (E) Asp Asp Gly (G) Pro, Ala Ala His (H) Asn, Gln, Lys, Arg ArgIle (I) Leu, Val, Met, Ala, Leu Phe, Norleucine Leu (L) Norleucine, Ile,Val, Ile Met, Ala, Phe Lys (K) Arg, 1,4 Diamino- Arg butyric Acid, Gln,Asn Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro(P) Ala Gly Ser (S) Thr, Ala, Cys Thr Thr (T) Ser Ser Trp (W) Tyr, PheTyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Met, Leu, Phe, Leu Ala,Norleucine

[0068] In certain embodiments, conservative amino acid substitutionsalso encompass non-naturally occurring amino acid residues which aretypically incorporated by chemical peptide synthesis rather than bysynthesis in biological systems.

[0069] As noted in the foregoing section “Definition of Terms,”naturally occurring residues may be divided into classes based on commonsidechain properties that may be useful for modifications of sequence.For example, non-conservative substitutions may involve the exchange ofa member of one of these classes for a member from another class. Suchsubstituted residues may be introduced into regions of the peptide thatare homologous with non-human orthologs, or into the non-homologousregions of the molecule. In addition, one may also make modificationsusing P or G for the purpose of influencing chain orientation.

[0070] In making such modifications, the hydropathic index of aminoacids may be considered. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and charge characteristics,these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

[0071] The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art.Kyte et al., J. Mol. Biol., 157: 105-131 (1982). It is known thatcertain amino acids may be substituted for other amino acids having asimilar hydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

[0072] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity. Thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsimmunogenicity and antigenicity, i.e., with a biological property of theprotein.

[0073] The following hydrophilicity values have been assigned to aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine(−2.5); tryptophan (−3.4). In making changes based upon similarhydrophilicity values, the substitution of amino acids whosehydrophilicity values are within ±2 is preferred, those which are within±1 are particularly preferred, and those within ±0.5 are even moreparticularly preferred. One may also identify epitopes from primaryamino acid sequences on the basis of hydrophilicity. These regions arealso referred to as “epitopic core regions.”

[0074] A skilled artisan will be able to determine suitable variants ofthe polypeptide as set forth in the foregoing sequences using well knowntechniques. For identifying suitable areas of the molecule that may bechanged without destroying activity, one skilled in the art may targetareas not believed to be important for activity. For example, whensimilar polypeptides with similar activities from the same species orfrom other species are known, one skilled in the art may compare theamino acid sequence of a peptide to similar peptides. With such acomparison, one can identify residues and portions of the molecules thatare conserved among similar polypeptides. It will be appreciated thatchanges in areas of a peptide that are not conserved relative to suchsimilar peptides would be less likely to adversely affect the biologicalactivity and/or structure of the peptide. One skilled in the art wouldalso know that, even in relatively conserved regions, one may substitutechemically similar amino acids for the naturally occurring residueswhile retaining activity (conservative amino acid residuesubstitutions). Therefore, even areas that may be important forbiological activity or for structure may be subject to conservativeamino acid substitutions without destroying the biological activity orwithout adversely affecting the peptide structure.

[0075] Additionally, one skilled in the art can reviewstructure-function studies identifying residues in similar peptides thatare important for activity or structure. In view of such a comparison,one can predict the importance of amino acid residues in a peptide thatcorrespond to amino acid residues that are important for activity orstructure in similar peptides. One skilled in the art may opt forchemically similar amino acid substitutions for such predicted importantamino acid residues of the peptides.

[0076] One skilled in the art can also analyze the three-dimensionalstructure and amino acid sequence in relation to that structure insimilar polypeptides. In view of that information, one skilled in theart may predict the alignment of amino acid residues of a peptide withrespect to its three dimensional structure. One skilled in the art maychoose not to make radical changes to amino acid residues predicted tobe on the surface of the protein, since such residues may be involved inimportant interactions with other molecules. Moreover, one skilled inthe art may generate test variants containing a single amino acidsubstitution at each desired amino acid residue. The variants can thenbe screened using activity assays know to those skilled in the art. Suchdata could be used to gather information about suitable variants. Forexample, if one discovered that a change to a particular amino acidresidue resulted in destroyed, undesirably reduced, or unsuitableactivity, variants with such a change would be avoided. In other words,based on information gathered from such routine experiments, one skilledin the art can readily determine the amino acids where furthersubstitutions should be avoided either alone or in combination withother mutations.

[0077] A number of scientific publications have been devoted to theprediction of secondary structure. See Moult J., Curr. Op. in Biotech.,7(4): 422-427 (1996), Chou et al. Biochemistry, 13(2): 222-245 (1974);Chou et al., Biochemistry, 113(2): 211-222 (1974); Chou et al., Adv.Enzymol. Relat. Areas Mol. Biol., 47: 45-148 (1978); Chou et al., Ann.Rev. Biochem., 47: 251-276 and Chou et al. Biophys. J., 26: 367-384(1979). Moreover, computer programs are currently available to assistwith predicting secondary structure. One method of predicting secondarystructure is based upon homology modeling. For example, two polypeptidesor proteins which have a sequence identity of greater than 30%, orsimilarity greater than 40% often have similar structural topologies.The recent growth of the protein structural data base (PDB) has providedenhanced predictability of secondary structure, including the potentialnumber of folds within a polypeptide's or protein's structure. See Holmet al., Nucl. Acid. Res., 27(1): 244-247 (1999). It has been suggested(Brenner et al., Curr. Op. Struct. Biol., 7(3): 369-376 (1997)) thatthere are a limited number of folds in a given polypeptide or proteinand that once a critical number of structures have been resolved,structural prediction will gain dramatically in accuracy.

[0078] Additional methods of predicting secondary structure include“threading” (Jones, D., Curr. Opin. Struct. Biol., 7(3): 377-87 (1997);Sippl et al., Structure, 4(1): 15-9 (1996)), “profile analysis” (Bowieet al., Science, 253: 164-170 (1991); Gribskov et al., Meth. Enzym.,183: 146-159 (1990); Gribskov et al., Proc. Nat. Acad. Sci., 84(13):4355-8 (1987)), and “evolutionary linkage” (See Home, supra, andBrenner, supra).

[0079] Vehicles. This invention requires the presence of at least onevehicle (F¹) attached to a peptide through the N-terminus, C-terminus ora sidechain of one of the amino acid residues. Multiple vehicles mayalso be used; e.g., Fc's at each terminus or an Fc at a terminus and aPEG group at the other terminus or a sidechain.

[0080] An Fc domain is the preferred vehicle. The Fc domain may be fusedto the N or C termini of the peptides or at both the N and C termini.Fusion to the N terminus is preferred.

[0081] As noted above, Fc variants are suitable vehicles within thescope of this invention. A native Fc may be extensively modified to forman Fc variant in accordance with this invention, provided binding to thesalvage receptor is maintained; see, for example WO 97/34631 and WO96/32478. In such Fc variants, one may remove one or more sites of anative Fc that provide structural features or functional activity notrequired by the fusion molecules of this invention. One may remove thesesites by, for example, substituting or deleting residues, insertingresidues into the site, or truncating portions containing the site. Theinserted or substituted residues may also be altered amino acids, suchas peptidomimetics or D-amino acids. Fc variants may be desirable for anumber of reasons, several of which are described below. Exemplary Fcvariants include molecules and sequences in which:

[0082] 1. Sites involved in disulfide bond formation are removed. Suchremoval may avoid reaction with other cysteine-containing proteinspresent in the host cell used to produce the molecules of the invention.For this purpose, the cysteine-containing segment at the N-terminus maybe truncated or cysteine residues may be deleted or substituted withother amino acids (e.g., alanyl, seryl). In particular, one may truncatethe N-terminal 20-amino acid segment of SEQ ID NO: 2 or delete orsubstitute the cysteine residues at positions 7 and 10 of SEQ ID NO: 2.Even when cysteine residues are removed, the single chain Fc domains canstill form a dimeric Fc domain that is held together non-covalently.

[0083] 2. A native Fc is modified to make it more compatible with aselected host cell. For example, one may remove the PA sequence near theN-terminus of a typical native Fc, which may be recognized by adigestive enzyme in E. coli such as proline iminopeptidase. One may alsoadd an N-terminal methionine residue, especially when the molecule isexpressed recombinantly in a bacterial cell such as E. coli. The Fcdomain of SEQ ID NO: 2 is one such Fc variant.

[0084] 3. A portion of the N-terminus of a native Fc is removed toprevent N-terminal heterogeneity when expressed in a selected host cell.For this purpose, one may delete any of the first 20 amino acid residuesat the N-terminus, particularly those at positions 1, 2, 3, 4 and 5.

[0085] 4. One or more glycosylation sites are removed. Residues that aretypically glycosylated (e.g., asparagine) may confer cytolytic response.Such residues may be deleted or substituted with unglycosylated residues(e.g., alanine).

[0086] 5. Sites involved in interaction with complement, such as the C1qbinding site, are removed. For example, one may delete or substitute theEKK sequence of human IgG1. Complement recruitment may not beadvantageous for the molecules of this invention and so may be avoidedwith such an Fc variant.

[0087] 6. Sites are removed that affect binding to Fc receptors otherthan a salvage receptor. A native Fc may have sites for interaction withcertain white blood cells that are not required for the fusion moleculesof the present invention and so may be removed.

[0088] 7. The ADCC site is removed. ADCC sites are known in the art;see, for example, Molec. Immunol. 29 (5): 633-9 (1992) with regard toADCC sites in IgG1. These sites, as well, are not required for thefusion molecules of the present invention and so may be removed.

[0089] 8. When the native Fc is derived from a non-human antibody, thenative Fc may be humanized. Typically, to humanize a native Fc, one willsubstitute selected residues in the non-human native Fc with residuesthat are normally found in human native Fc. Techniques for antibodyhumanization are well known in the art.

[0090] Preferred Fc variants include the following. In SEQ ID NO: 2(FIG. 4) the leucine at position 15 may be substituted with glutamate;the glutamate at position 99, with alanine; and the lysines at positions101 and 103, with alanines. In addition, one or more tyrosine residuescan be replaced by phenyalanine residues.

[0091] An alternative vehicle would be a protein, polypeptide, peptide,antibody, antibody fragment, or small molecule (e.g., a peptidomimeticcompound) capable of binding to a salvage receptor. For example, onecould use as a vehicle a polypeptide as described in U.S. Pat. No.5,739,277, issued Apr. 14, 1998 to Presta et al. Peptides could also beselected by phage display or RNA-peptide screening for binding to theFcRn salvage receptor. Such salvage receptor-binding compounds are alsoincluded within the meaning of “vehicle” and are within the scope ofthis invention. Such vehicles should be selected for increased half-life(e.g., by avoiding sequences recognized by proteases) and decreasedimmunogenicity (e.g., by favoring non-immunogenic sequences, asdiscovered in antibody humanization).

[0092] As noted above, polymer vehicles may also be used for F¹. Variousmeans for attaching chemical moieties useful as vehicles are currentlyavailable, see e.g., Patent Cooperation Treaty (“PCT”) InternationalPublication No. WO 96/11953, entitled “N-Terminally Chemically ModifiedProtein Compositions and Methods,” herein incorporated by reference inits entirety. This PCT publication discloses, among other things, theselective attachment of water soluble polymers to the N-terminus ofproteins.

[0093] A preferred polymer vehicle is polyethylene glycol (PEG). The PEGgroup may be of any convenient molecular weight and may be linear orbranched. The average molecular weight of the PEG will preferably rangefrom about 2 kiloDalton (“kD”) to about 100 kD, more preferably fromabout 5 kD to about 50 kD, most preferably from about 5 kD to about 10kD. The PEG groups will generally be attached to the compounds of theinvention via acylation or reductive alkylation through a reactive groupon the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to areactive group on the inventive compound (e.g., an aldehyde, amino, orester group).

[0094] A useful strategy for the PEGylation of synthetic peptidesconsists of combining, through forming a conjugate linkage in solution,a peptide and a PEG moiety, each bearing a special functionality that ismutually reactive toward the other. The peptides can be easily preparedwith conventional solid phase synthesis (see, for example, FIGS. 5 and 6and the accompanying text herein). The peptides are “preactivated” withan appropriate functional group at a specific site. The precursors arepurified and fully characterized prior to reacting with the PEG moiety.Ligation of the peptide with PEG usually takes place in aqueous phaseand can be easily monitored by reverse phase analytical HPLC. ThePEGylated peptides can be easily purified by preparative HPLC andcharacterized by analytical HPLC, amino acid analysis and laserdesorption mass spectrometry.

[0095] Polysaccharide polymers are another type of water soluble polymerwhich may be used for protein modification. Dextrans are polysaccharidepolymers comprised of individual subunits of glucose predominantlylinked by α1-6 linkages. The dextran itself is available in manymolecular weight ranges, and is readily available in molecular weightsfrom about 1 kD to about 70 kD. Dextran is a suitable water solublepolymer for use in the present invention as a vehicle by itself or incombination with another vehicle (e.g., Fc). See, for example, WO96/11953 and WO 96/05309. The use of dextran conjugated to therapeuticor diagnostic immunoglobulins has been reported; see, for example,European Patent Publication No. 0 315 456, which is hereby incorporatedby reference in its entirety. Dextran of about 1 kD to about 20 kD ispreferred when dextran is used as a vehicle in accordance with thepresent invention.

[0096] Linkers. Any “linker” group is optional. When present, itschemical structure is not critical, since it serves primarily as aspacer. The linker is preferably made up of amino acids linked togetherby peptide bonds. Thus, in preferred embodiments, the linker is made upof from 1 to 20 amino acids linked by peptide bonds, wherein the aminoacids are selected from the 20 naturally occurring amino acids. Some ofthese amino acids may be glycosylated, as is well understood by those inthe art. In a more preferred embodiment, the 1 to 20 amino acids areselected from glycine, alanine, proline, asparagine, glutamine, andlysine. Even more preferably, a linker is made up of a majority of aminoacids that are sterically unhindered, such as glycine and alanine. Thus,preferred linkers are polyglycines (particularly (Gly)₄, (Gly)₅),poly(Gly-Ala), and polyalanines. Other specific examples of linkers are:(Gly)₃Lys(Gly)₄ (SEQ ID NO:3); (Gly)₃AsnGlySer(Gly)₂ (SEQ ID NO:4);(Gly)₃Cys(Gly)₄ (SEQ ID NO:5); and GlyProAsnGlyGly (SEQ ID NO:6).

[0097] To explain the above nomenclature, for example, (Gly)₃Lys(Gly)₄means Gly-Gly-Gly-Lys-Gly-Gly-Gly-Gly (SEQ ID NO: 3). Single amino acidlinkers (e.g., -Ala- or -Gly-) are preferred linkers, and combinationsof Gly and Ala are also preferred. The linkers shown here are exemplary;linkers within the scope of this invention may be much longer and mayinclude other residues.

[0098] Non-peptide linkers are also possible. For example, alkyl linkerssuch as —NH—(CH₂)_(s)—C(O)—, wherein s=2-20 could be used. These alkyllinkers may further be substituted by any non-sterically hindering groupsuch as lower alkyl (e.g., C₁-C₆) lower acyl, halogen (e.g., Cl, Br),CN, NH₂, phenyl, etc. An exemplary non-peptide linker is a PEG linker,

[0099] wherein n is such that the linker has a molecular weight of 100to 5000 kD, preferably 100 to 500 kD. The peptide linkers may be alteredto form derivatives in the same manner as described above.

[0100] Derivatives. The inventors also contemplate derivatizing thepeptide and/or vehicle portion of the compounds. Such derivatives mayimprove the solubility, absorption, biological half life, and the likeof the compounds. The moieties may alternatively eliminate or attenuateany undesirable side-effect of the compounds and the like. Exemplaryderivatives include compounds in which:

[0101] 1. The compound or some portion thereof is cyclic. For example,the peptide portion may be modified to contain two or more Cys residues(e.g., in the linker), which could cyclize by disulfide bond formation.

[0102] 2. The compound is cross-linked or is rendered capable ofcross-linking between molecules. For example, the peptide portion may bemodified to contain one Cys residue and thereby be able to form anintermolecular disulfide bond with a like molecule. The compound mayalso be cross-linked through its C-terminus, as in the molecule shownbelow.

[0103] 3. One or more peptidyl [—C(O)NR—] linkages (bonds) is replacedby a non-peptidyl linkage. Exemplary non-peptidyl linkages are—CH₂-carbamate [—CH₂—OC(O)NR—], phosphonate, —CH₂-sulfonamide[—CH₂—S(O)₂NR—], urea [—NHC(O)NH—], —CH₂-secondary amine, and alkylatedpeptide [—C(O)NR⁶— wherein R⁶ is lower alkyl].

[0104]4. The N-terminus is derivatized. Typically, the N-terminus may beacylated or modified to a substituted amine. Exemplary N-terminalderivative groups include —NRR¹ (other than —NH₂), —NRC(O)R¹,—NRC(O)OR¹, —NRS(O)₂R¹, —NHC(O)NHR¹, succinimide, orbenzyloxycarbonyl-NH— (CBZ-NH—), wherein R and R¹ are each independentlyhydrogen or lower alkyl and wherein the phenyl ring may be substitutedwith 1 to 3 substituents selected from the group consisting of C₁-C₄alkyl, C₁-C₄ alkoxy, chloro, and bromo.

[0105] 5. The free C-terminus is derivatized. Typically, the C-terminusis esterified or amidated. Exemplary C-terminal derivative groupsinclude, for example, —C(O)R² wherein R² is lower alkoxy or —NR³R⁴wherein R³ and R⁴ are independently hydrogen or C₁-C₈ alkyl (preferablyC₁-C₄ alkyl).

[0106] 6. A disulfide bond is replaced with another, preferably morestable, cross-linking moiety (e.g., an alkylene). See, e.g., Bhatnagaret al. (1996), J. Med. Chem. 39: 3814-9; Alberts et al. (1993)Thirteenth Am. Pep. Symp., 357-9.

[0107] 7. One or more individual amino acid residues is modified.Various derivatizing agents are known to react specifically withselected sidechains or terminal residues, as described in detail below.

[0108] Lysinyl residues and amino terminal residues may be reacted withsuccinic or other carboxylic acid anhydrides, which reverse the chargeof the lysinyl residues. Other suitable reagents for derivatizingalpha-amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride;trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; andtransaminase-catalyzed reaction with glyoxylate.

[0109] Arginyl residues may be modified by reaction with any one orcombination of several conventional reagents, including phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization ofarginyl residues requires that the reaction be performed in alkalineconditions because of the high pKa of the guanidine functional group.Furthermore, these reagents may react with the groups of lysine as wellas the arginine epsilon-amino group.

[0110] Specific modification of tyrosyl residues has been studiedextensively, with particular interest in introducing spectral labelsinto tyrosyl residues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizole andtetranitromethane are used to form O-acetyl tyrosyl species and 3-nitroderivatives, respectively.

[0111] Carboxyl sidechain groups (aspartyl or glutamyl) may beselectively modified by reaction with carbodiimides (R′—N═C═N—R′) suchas 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

[0112] Glutaminyl and asparaginyl residues may be deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

[0113] Cysteinyl residues can be replaced by amino acid residues orother moieties either to eliminate disulfide bonding or, conversely, tostabilize cross-linking. See, e.g., Bhatnagar et al. (1996), J. Med.Chem. 39: 3814-9.

[0114] Derivatization with bifunctional agents is useful forcross-linking the peptides or their functional derivatives to awater-insoluble support matrix or to other macromolecular vehicles.Commonly used cross-linking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

[0115] Carbohydrate (oligosaccharide) groups may conveniently beattached to sites that are known to be glycosylation sites in proteins.Generally, O-linked oligosaccharides are attached to serine (Ser) orthreonine (Thr) residues while N-linked oligosaccharides are attached toasparagine (Asn) residues when they are part of the sequenceAsn-X-Ser/Thr, where X can be any amino acid except proline. X ispreferably one of the 19 naturally occurring amino acids other thanproline. The structures of N-linked and O-linked oligosaccharides andthe sugar residues found in each type are different. One type of sugarthat is commonly found on both is N-acetylneuraminic acid (referred toas sialic acid). Sialic acid is usually the terminal residue of bothN-linked and O-linked oligosaccharides and, by virtue of its negativecharge, may confer acidic properties to the glycosylated compound. Suchsite(s) may be incorporated in the linker of the compounds of thisinvention and are preferably glycosylated by a cell during recombinantproduction of the polypeptide compounds (e.g., in mammalian cells suchas CHO, BHK, COS). However, such sites may further be glycosylated bysynthetic or semi-synthetic procedures known in the art.

[0116] Other possible modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, oxidation of the sulfur atom in Cys, methylation of thealpha-amino groups of lysine, arginine, and histidine side chains.Creighton, Proteins: Structure and Molecule Properties (W. H. Freeman &Co., San Francisco), pp. 79-86 (1983).

[0117] Compounds of the present invention may be changed at the DNAlevel, as well. The DNA sequence of any portion of the compound may bechanged to codons more compatible with the chosen host cell. For E.coli, which is the preferred host cell, optimized codons are known inthe art. Codons may be substituted to eliminate restriction sites or toinclude silent restriction sites, which may aid in processing of the DNAin the selected host cell. The vehicle, linker and peptide DNA sequencesmay be modified to include any of the foregoing sequence changes.

[0118] Methods of Making

[0119] The compounds of this invention largely may be made intransformed host cells using recombinant DNA techniques. To do so, arecombinant DNA molecule coding for the peptide is prepared. Methods ofpreparing such DNA molecules are well known in the art. For instance,sequences coding for the peptides could be excised from DNA usingsuitable restriction enzymes. Alternatively, the DNA molecule could besynthesized using chemical synthesis techniques, such as thephosphoramidate method. Also, a combination of these techniques could beused.

[0120] The invention also includes a vector capable of expressing thepeptides in an appropriate host. The vector comprises the DNA moleculethat codes for the peptides operatively linked to appropriate expressioncontrol sequences. Methods of effecting this operative linking, eitherbefore or after the DNA molecule is inserted into the vector, are wellknown. Expression control sequences include promoters, activators,enhancers, operators, ribosomal binding sites, start signals, stopsignals, cap signals, polyadenylation signals, and other signalsinvolved with the control of transcription or translation.

[0121] The resulting vector having the DNA molecule thereon is used totransform an appropriate host. This transformation may be performedusing methods well known in the art.

[0122] Any of a large number of available and well-known host cells maybe used in the practice of this invention. The selection of a particularhost is dependent upon a number of factors recognized by the art. Theseinclude, for example, compatibility with the chosen expression vector,toxicity of the peptides encoded by the DNA molecule, rate oftransformation, ease of recovery of the peptides, expressioncharacteristics, bio-safety and costs. A balance of these factors mustbe struck with the understanding that not all hosts may be equallyeffective for the expression of a particular DNA sequence. Within thesegeneral guidelines, useful microbial hosts include bacteria (such as E.coli sp.), yeast (such as Saccharomyces sp.) and other fungi, insects,plants, mammalian (including human) cells in culture, or other hostsknown in the art.

[0123] Next, the transformed host is cultured and purified. Host cellsmay be cultured under conventional fermentation conditions so that thedesired compounds are expressed. Such fermentation conditions are wellknown in the art. Finally, the peptides are purified from culture bymethods well known in the art.

[0124] The compounds may also be made by synthetic methods. For example,solid phase synthesis techniques may be used. Suitable techniques arewell known in the art, and include those described in Merrifield (1973),Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.);Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985),Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid PhasePeptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), TheProteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins(3rd ed.) 2: 257-527. Solid phase synthesis is the preferred techniqueof making individual peptides since it is the most cost-effective methodof making small peptides.

[0125] Compounds that contain derivatized peptides or which containnon-peptide groups may be synthesized by well-known organic chemistrytechniques.

[0126] Uses of the Compounds

[0127] The compounds of this invention have pharmacologic activityresulting from their Apo-AI amphipathic helix peptide-mimetic activity.

[0128] Agonists or mimetics of Apo-AI amphipathic helix peptide areuseful in treating:

[0129] hypercholesterolemia

[0130] viral infection, particularly HSV and HIV infection; and thelike.

[0131] Pharmaceutical Compositions

[0132] In General. The present invention also provides methods of usingpharmaceutical compositions of the inventive compounds. Suchpharmaceutical compositions may be for administration for injection, orfor oral, pulmonary, nasal, transdermal or other forms ofadministration. In general, the invention encompasses pharmaceuticalcompositions comprising effective amounts of a compound of the inventiontogether with pharmaceutically acceptable diluents, preservatives,solubilizers, emulsifiers, adjuvants and/or carriers. Such compositionsinclude diluents of various buffer content (e.g., Tris-HCl, acetate,phosphate), pH and ionic strength; additives such as detergents andsolubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants(e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g.,Thimersol, benzyl alcohol) and bulking substances (e.g., lactose,mannitol); incorporation of the material into particulate preparationsof polymeric compounds such as polylactic acid, polyglycolic acid, etc.or into liposomes. Hyaluronic acid may also be used, and this may havethe effect of promoting sustained duration in the circulation. Suchcompositions may influence the physical state, stability, rate of invivo release, and rate of in vivo clearance of the present proteins andderivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed.(1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which areherein incorporated by reference in their entirety. The compositions maybe prepared in liquid form, or may be in dried powder, such aslyophilized form. Implantable sustained release formulations are alsocontemplated, as are transdermal formulations.

[0133] Oral dosage forms. Contemplated for use herein are oral soliddosage forms, which are described generally in Chapter 89 of Remington'sPharmaceutical Sciences (1990), 18th Ed., Mack Publishing Co. Easton Pa.18042, which is herein incorporated by reference in its entirety. Soliddosage forms include tablets, capsules, pills, troches or lozenges,cachets or pellets. Also, liposomal or proteinoid encapsulation may beused to formulate the present compositions (as, for example, proteinoidmicrospheres reported in U.S. Pat. No. 4,925,673). Liposomalencapsulation may be used and the liposomes may be derivatized withvarious polymers (e.g., U.S. Pat. No. 5,013,556). A description ofpossible solid dosage forms for the therapeutic is given in Chapter 10of Marshall, K., Modern Pharmaceutics (1979), edited by G. S. Banker andC. T. Rhodes, herein incorporated by reference in its entirety. Ingeneral, the formulation will include the inventive compound, and inertingredients which allow for protection against the stomach environment,and release of the biologically active material in the intestine.

[0134] Also specifically contemplated are oral dosage forms of the aboveinventive compounds. If necessary, the compounds may be chemicallymodified so that oral delivery is efficacious. Generally, the chemicalmodification contemplated is the attachment of at least one moiety tothe compound molecule itself, where said moiety permits (a) inhibitionof proteolysis; and (b) uptake into the blood stream from the stomach orintestine. Also desired is the increase in overall stability of thecompound and increase in circulation time in the body. Moieties usefulas covalently attached vehicles in this invention may also be used forthis purpose. Examples of such moieties include: PEG, copolymers ofethylene glycol and propylene glycol, carboxymethyl cellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. See, forexample, Abuchowski and Davis, Soluble Polymer-Enzyme Adducts, Enzymesas Drugs (1981), Hocenberg and Roberts, eds., Wiley-Interscience, NewYork, N.Y., pp. 367-83; Newmark, et al. (1982), J. Appl. Biochem.4:185-9. Other polymers that could be used are poly-1,3-dioxolane andpoly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as indicatedabove, are PEG moieties.

[0135] For oral delivery dosage forms, it is also possible to use a saltof a modified aliphatic amino acid, such as sodiumN-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), as a carrier to enhanceabsorption of the therapeutic compounds of this invention. The clinicalefficacy of a heparin formulation using SNAC has been demonstrated in aPhase II trial conducted by Emisphere Technologies. See U.S. Pat. No.5,792,451, “Oral drug delivery composition and methods”.

[0136] The compounds of this invention can be included in theformulation as fine multiparticulates in the form of granules or pelletsof particle size about 1 mm. The formulation of the material for capsuleadministration could also be as a powder, lightly compressed plugs oreven as tablets. The therapeutic could be prepared by compression.

[0137] Colorants and flavoring agents may all be included. For example,the protein (or derivative) may be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

[0138] One may dilute or increase the volume of the compound of theinvention with an inert material. These diluents could includecarbohydrates, especially mannitol, α-lactose, anhydrous lactose,cellulose, sucrose, modified dextrans and starch. Certain inorganicsalts may also be used as fillers including calcium triphosphate,magnesium carbonate and sodium chloride. Some commercially availablediluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

[0139] Disintegrants may be included in the formulation of thetherapeutic into a solid dosage form. Materials used as disintegrantsinclude but are not limited to starch including the commercialdisintegrant based on starch, Explotab. Sodium starch glycolate,Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodiumalginate, gelatin, orange peel, acid carboxymethyl cellulose, naturalsponge and bentonite may all be used. Another form of the disintegrantsare the insoluble cationic exchange resins. Powdered gums may be used asdisintegrants and as binders and these can include powdered gums such asagar, Karaya or tragacanth. Alginic acid and its sodium salt are alsouseful as disintegrants.

[0140] Binders may be used to hold the therapeutic agent together toform a hard tablet and include materials from natural products such asacacia, tragacanth, starch and gelatin. Others include methyl cellulose(MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

[0141] An antifrictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantsmay also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, Carbowax 4000and 6000.

[0142] Glidants that might improve the flow properties of the drugduring formulation and to aid rearrangement during compression might beadded. The glidants may include starch, talc, pyrogenic silica andhydrated silicoaluminate.

[0143] To aid dissolution of the compound of this invention into theaqueous environment a surfactant might be added as a wetting agent.Surfactants may include anionic detergents such as sodium laurylsulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.Cationic detergents might be used and could include benzalkoniumchloride or benzethonium chloride. The list of potential nonionicdetergents that could be included in the formulation as surfactants arelauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenatedcastor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65and 80, sucrose fatty acid ester, methyl cellulose and carboxymethylcellulose. These surfactants could be present in the formulation of theprotein or derivative either alone or as a mixture in different ratios.

[0144] Additives may also be included in the formulation to enhanceuptake of the compound. Additives potentially having this property arefor instance the fatty acids oleic acid, linoleic acid and linolenicacid.

[0145] Controlled release formulation may be desirable. The compound ofthis invention could be incorporated into an inert matrix which permitsrelease by either diffusion or leaching mechanisms e.g., gums. Slowlydegenerating matrices may also be incorporated into the formulation,e.g., alginates, polysaccharides. Another form of a controlled releaseof the compounds of this invention is by a method based on the Orostherapeutic system (Alza Corp.), i.e., the drug is enclosed in asemipermeable membrane which allows water to enter and push drug outthrough a single small opening due to osmotic effects. Some entericcoatings also have a delayed release effect.

[0146] Other coatings may be used for the formulation. These include avariety of sugars which could be applied in a coating pan. Thetherapeutic agent could also be given in a film coated tablet and thematerials used in this instance are divided into 2 groups. The first arethe nonenteric materials and include methyl cellulose, ethyl cellulose,hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropylcellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methylcellulose, providone and the polyethylene glycols. The second groupconsists of the enteric materials that are commonly esters of phthalicacid.

[0147] A mix of materials might be used to provide the optimum filmcoating. Film coating may be carried out in a pan coater or in afluidized bed or by compression coating.

[0148] Pulmonary delivery forms. Also contemplated herein is pulmonarydelivery of the present protein (or derivatives thereof). The protein(or derivative) is delivered to the lungs of a mammal while inhaling andtraverses across the lung epithelial lining to the blood stream. (Otherreports of this include Adjei et al., Pharma. Res. (1990) 7: 565-9;Adjei et al. (1990), Internatl. J. Pharmaceutics 63: 135-44 (leuprolideacetate); Braquet et al. (1989), J. Cardiovasc. Pharmacol. 13 (suppl.5):s.143-146 (endothelin-1); Hubbard et al. (1989), Annals Int. Med. 3:206-12 (α1-antitrypsin); Smith et al. (1989), J. Clin. Invest. 84:1145-6 (α1-proteinase); Oswein et al. (March 1990), “Aerosolization ofProteins”, Proc. Symp. Resp. Drug Delivery II, Keystone, Colorado(recombinant human growth hormone); Debs et al. (1988), J. Immunol. 140:3482-8 (interferon-γ and tumor necrosis factor α) and Platz et al., U.S.Pat. No. 5,284,656 (granulocyte colony stimulating factor).

[0149] Contemplated for use in the practice of this invention are a widerange of mechanical devices designed for pulmonary delivery oftherapeutic products, including but not limited to nebulizers, metereddose inhalers, and powder inhalers, all of which are familiar to thoseskilled in the art. Some specific examples of commercially availabledevices suitable for the practice of this invention are the Ultraventnebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the AcornII nebulizer, manufactured by Marquest Medical Products, Englewood,Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc.,Research Triangle Park, N.C.; and the Spinhaler powder inhaler,manufactured by Fisons Corp., Bedford, Mass.

[0150] All such devices require the use of formulations suitable for thedispensing of the inventive compound. Typically, each formulation isspecific to the type of device employed and may involve the use of anappropriate propellant material, in addition to diluents, adjuvantsand/or carriers useful in therapy.

[0151] The inventive compound should most advantageously be prepared inparticulate form with an average particle size of less than 10 μm (ormicrons), most preferably 0.5 to 5 μm, for most effective delivery tothe distal lung.

[0152] Pharmaceutically acceptable carriers include carbohydrates suchas trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Otheringredients for use in formulations may include DPPC, DOPE, DSPC andDOPC. Natural or synthetic surfactants may be used. PEG may be used(even apart from its use in derivatizing the protein or analog).Dextrans, such as cyclodextran, may be used. Bile salts and otherrelated enhancers may be used. Cellulose and cellulose derivatives maybe used. Amino acids may be used, such as use in a buffer formulation.

[0153] Also, the use of liposomes, microcapsules or microspheres,inclusion complexes, or other types of carriers is contemplated.

[0154] Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise the inventive compound dissolved inwater at a concentration of about 0.1 to 25 mg of biologically activeprotein per mL of solution. The formulation may also include a bufferand a simple sugar (e.g., for protein stabilization and regulation ofosmotic pressure). The nebulizer formulation may also contain asurfactant, to reduce or prevent surface induced aggregation of theprotein caused by atomization of the solution in forming the aerosol.

[0155] Formulations for use with a metered-dose inhaler device willgenerally comprise a finely divided powder containing the inventivecompound suspended in a propellant with the aid of a surfactant. Thepropellant may be any conventional material employed for this purpose,such as a chlorofluorocarbon, a hydrochlorofluorocarbon, ahydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethanol, and1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactantsinclude sorbitan trioleate and soya lecithin. Oleic acid may also beuseful as a surfactant.

[0156] Formulations for dispensing from a powder inhaler device willcomprise a finely divided dry powder containing the inventive compoundand may also include a bulking agent, such as lactose, sorbitol,sucrose, mannitol, trehalose, or xylitol in amounts which facilitatedispersal of the powder from the device, e.g., 50 to 90% by weight ofthe formulation.

[0157] Nasal delivery forms. Nasal delivery of the inventive compound isalso contemplated. Nasal delivery allows the passage of the protein tothe blood stream directly after administering the therapeutic product tothe nose, without the necessity for deposition of the product in thelung. Formulations for nasal delivery include those with dextran orcyclodextran. Delivery via transport across other mucous membranes isalso contemplated.

[0158] Dosages. The dosage regimen involved in a method for treating theabove-described conditions will be determined by the attendingphysician, considering various factors which modify the action of drugs,e.g. the age, condition, body weight, sex and diet of the patient, theseverity of any infection, time of administration and other clinicalfactors. Generally, the daily regimen should be in the range of 0.1-1000micrograms of the inventive compound per kilogram of body weight,preferably 0.1-150 micrograms per kilogram.

[0159] Specific Preferred Embodiments

[0160] The inventors have determined preferred structures for thepreferred peptides listed in Table 2 below. The symbol “Λ” may be any ofthe linkers described herein or may simply represent a normal peptidebond (i.e., so that no linker is present). Tandem repeats and linkersare shown separated by dashes for clarity. TABLE 2 Preferred embodimentsSEQ ID Sequence/structure NO: Asp Trp Leu Lys Ala Phe Tyr Asp Lys ValAla 8 Glu Lys Leu Lys Glu Ala Phe-Λ-F¹ F¹-Λ-Asp Trp Leu Lys Ala Phe TyrAsp Lys Val 9 Ala Glu Lys Leu Lys Glu Ala Phe Asp Trp Leu Lys Ala PheTyr Asp Lys Val Ala 10 Glu Lys Leu Lys Glu Ala Phe-Λ-Asp Trp Leu Lys AlaPhe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu Ala Phe-Λ-F¹ F¹-Λ-Asp TrpLeu Lys Ala Phe Tyr Asp Lys Val 11 Ala Glu Lys Leu Lys Glu Ala Phe-Λ-AspTrp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu Ala Phe

[0161] “F¹” is an Fc domain as defined previously herein. In addition tothose listed in Table 2, the inventors further contemplate heterodimersin which each strand of an Fc dimer is linked to a different peptidesequence; for example, wherein each Fc is linked to a different sequenceselected from Table 1.

[0162] All of the compounds of this invention can be prepared by methodsdescribed in PCT appl. no. WO 99/25044.

[0163] The invention now being fully described, it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made thereto, without departing from the spirit and scope of theinvention as set forth herein.

What is claimed is:
 1. A composition of matter of the formula(A¹)_(a)-F¹-(A²)_(b) and multimers thereof, wherein: F¹ is a vehicle; A¹and A² are each independently selected from -(L¹)_(c)-P¹,-(L¹)_(c)-P¹-(L²)_(d)-P², -(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³, and-(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³-(L⁴)_(f)-P⁴ P¹, P², P³, and P⁴ areeach independently sequences of Apo-AI amphipathic helix peptide orApo-AI amphipathic helix peptide-mimetic domains; L¹, L₂, L³, and L⁴ areeach independently linkers; and a, b, c, d, e, and f are eachindependently 0 or 1, provided that at least one of a and b is
 1. 2. Thecomposition of matter of claim 1 of the formulae A¹-F¹orF¹-A².
 3. Thecomposition of matter of claim 1 of the formula F¹-(L¹)_(c)-P¹.
 4. Thecomposition of matter of claim 1 of the formulaF¹-(L¹)_(c)-P¹-(L²)_(d)-P².
 5. The composition of matter of claim 1,wherein F¹ is an Fc domain.
 6. The composition of matter of claim 1wherein F¹ is an IgG Fc domain.
 7. The composition of matter of claim 1wherein F¹ is an IgG1 Fc domain.
 8. The composition of matter of claim 1wherein F¹ comprises the sequence of SEQ ID NO:
 2. 9. The composition ofmatter of claim 1 wherein the Apo-AI amphipathic helix peptide or Apo-AIamphipathic helix peptide-mimetic domain sequence is of the formula AspTrp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu Ala Phe (SEQID NO: 7)
 10. The composition of matter of claim 9, wherein F¹ is an Fcdomain.
 11. The composition of matter of claim 9, wherein F¹ is an IgGFc domain.
 12. The composition of matter of claim 11, wherein F¹ is anIgG1 Fc domain.
 13. The composition of matter of claim 5, having asequence selected from Table 2 (SEQ ID NOS: 8 to 11).
 14. A DNA encodinga composition of matter of claim
 5. 15. A DNA encoding a composition ofmatter of claim
 10. 16. An expression vector comprising the DNA of claim14.
 17. An expression vector comprising the DNA of claim
 15. 18. A hostcell comprising the expression vector of claim
 16. 19. A host cellcomprising the expression vector of claim
 17. 20. The cell of claim 18,wherein the cell is an E. coli cell.
 21. The cell of claim 19, whereinthe cell is an E. coli cell.
 22. A process for preparing a Apo-AIamphipathic helix peptide-mimetic compound, which comprises: a)selecting Apo-AI amphipathic helix peptide or at least one Apo-AIamphipathic helix peptide-mimetic peptide; and b) preparing apharmacologic agent comprising at least one Fc domain covalently linkedto at least one amino acid sequence of the selected peptide or peptidesfrom step a).
 23. The process of claim 22, wherein the peptide isselected from the SEQ ID NO:
 7. 24. The process of claim 22, wherein thepeptide is selected in a process comprising screening of a phage displaylibrary, an E. coli display library, a ribosomal library, an RNA-peptidelibrary, or a chemical peptide library.
 25. The process of claim 22,wherein the preparation of the pharmacologic agent is carried out by: a)preparing a gene construct comprising a nucleic acid sequence encodingthe selected peptide and a nucleic acid sequence encoding an Fc domain;and b) expressing the gene construct.
 26. The process of claim 25,wherein the gene construct is expressed in an E. coli cell.
 27. Theprocess of claim 22, wherein the selection of the peptide is carried outby a process comprising: a) preparing a gene construct comprising anucleic acid sequence encoding a first selected peptide and a nucleicacid sequence encoding an Fc domain; b) conducting a polymerase chainreaction using the gene construct and mutagenic primers, wherein i) afirst mutagenic primer comprises a nucleic acid sequence complementaryto a sequence at or near the 5′ end of a coding strand of the geneconstruct, and ii) a second mutagenic primer comprises a nucleic acidsequence complementary to the 3′ end of the noncoding strand of the geneconstruct.
 28. A method of treating hypercholesterolemia, whichcomprises administering a composition of matter of claim
 1. 29. A methodof treating viral infection, which comprises administering a compositionof matter of claim
 1. 30. The method of claim 29, wherein the viralinfection is an HSV infection.
 31. The method of claim 29, wherein theviral infection is an HIV infection.